1. Field of the Invention
This invention relates to methods for reducing the intra-ocular pressure of the eye by enhancing aqueous humor outflow, and to a method for screening compounds that reduce intra-ocular pressure.
2. Description of Related Art
In glaucoma, a leading cause of blindness, the optic nerve is damaged through a poorly-understood interaction of elevated intra-ocular pressure (IOP) and patient predisposition to the disease. In the most common form of glaucoma the trabecular meshwork (TM), which plays a critical role in regulation of aqueous humor outflow and intra-ocular pressure in both health and disease, is thought to be defective in such a manner that resistance to outflow and IOP both rise.
The anterior chamber of the eye is bathed with aqueous humor, formed continuously by the ciliary body. Aqueous humor moves by bulk flow from its site of production in the posterior chamber through the pupillary aperture and into the anterior chamber. It subsequently exits the anterior chamber via one of two routes.
The majority of outflow in the healthy human eve occurs at the anterior chamber angle, where aqueous humor passes through the trabecular meshwork and into the Canal of Schlemm, from where it joins the general venous drainage of the eye. A second outflow pathway is via the uveoscleral route, although this appears to be a minor (≈20%) pathway in the normal human eve. A homeostatic balance of aqueous humor production and drainage allows intra-ocular pressure to be maintained within narrow limits in the normal eye (Caprioli, J., Adler""s Physiology of the Eye: Clinical Application, W. M. Hart, ed. 9th Ed., 7:228-247, 1992; Hart, W. M., Adler""s Physiology of the Eye: Clinical Application, W. M. Hart, ed. 9th Ed., 8:248-267, 1992).
Production of aqueous humor occurs along the surface of the ciliary processes (pars plicata), which is covered by a double layer of epithelial cells consisting of a pigmented and non-pigmented layer situated with their apical surfaces juxtaposed. These function in tandem to produce transepithelial secretion of NaCl and water in movement from the blood to the aqueous humor. Evidence has been provided that Naxe2x80x94Kxe2x80x94Cl cotransport and the Na/K pump act in concert to bring about the vectorial transport (Dong, et al., Invest. Ophthalmol. Vis. Sci., 35:1660, 1994). The rate of aqueous humor production is quite high relative to other types of epithelia that function in vectorial transport of water and electrolytes. Thus, a drainage pathway that can accommodate this rate of fluid production is essential for maintenance of normal intra-ocular pressure. The aqueous humor production and drainage mechanisms work to replace the entire volume of aqueous every 100 minutes (Caprioli, J., supra).
It is well recognized that regulation of aqueous humor outflow through the trabecular meshwork is critically important for maintenance of an appropriate intra-ocular pressure, and that in disease states such as ocular hypertension and glaucoma, this regulation appears to be defective. For instance. U.S. Pat. No. 4,757,089 teaches a method for increasing aqueous humor outflow by topical or intracameral administration of ethacrynic acid, or an analog, to treat glaucoma. It is also known that ethacrynic acid increases water flux across the walls of perfused microvessels (Brandt, et al., Invest. Ophthalmol. Vis. Sci., 35(4[Suppl]): 1848, 1994) and inhibits Na+xe2x80x94K+xe2x80x942Clxe2x88x92 cotransport activity of avian erythrocytes (Palfrey, et al., Am. J. Physiol., 264:C1270-C1277, 1993), although the mechanisms by which these phenomena occur have not been elucidated. For instance, phenoxyacetic acids inhibit NaCl reabsorption in the thick ascending limb of the loop of Henle screening test, but its effect was exerted from both epithelial sides, rather than from the luminal side as with the class of loop diuretics, and it led to a depolarization of the membrane voltage. This effect is compatible with an inhibitory action at the level of mitochondrial ATP production rather than an inhibition of the Na+xe2x80x94K+xe2x80x942Clxe2x88x92 cotransporter.
In contrast to the current level of knowledge regarding cellular processes responsible for aqueous humor production by the ciliary body, relatively little is known about the cellular mechanisms in the trabecular meshwork that determine the rate of aqueous outflow. Pinocytotic vesicles are observed in the juxtacanalicular meshwork and the inner wall of Schlemm""s Canal. The function of these vesicles remains unknown, but some investigators have suggested that the bulk flow of aqueous humor through the meshwork cannot be accounted for by flow through the intercellular spaces and that these vesicles play a central role in outflow regulation. Evidence has been provided that cytoskeleton-mediated changes in trabecular meshwork cell shape modulate aqueous outflow (Erickson-Lamy and Nathanson, Invest. Ophthalmol. Vis. Sci., 33:2672-2678, 1992; Erickson-Lamy, Schroder, and Epstein, Invest. Opthalmol. Vis. Sci., 3263 1-2640. 1992). The extracellular matrix surrounding the trabeculae is thought to contribute to outflow resistance, perhaps by interactions with proteins contained in the aqueous humor (Freddo, T. F., Optometry Vis. Sci., 70:263-270, 1993). Indeed. abnormalities in this extracellular matrix may contribute to the increased outflow resistance seen in corticosteroid-induced glaucoma (Partridge, et al., Invest. Ophthalmol. Vis. Sci., 30:1843-1847, 1989; Polansky, et al., The Ocular Effects of Prostaglandins and Other Eicosanoids, Alan R. Liss, Inc., pp. 113-138, 1989). Investigators evaluating both normal physiology and drug effects have provided evidence that changes in cell shape (as distinct from cell volume) may be involved in outflow regulation (Erickson-Lamy and Nathanson, supra; Erickson-Lamy, Schroder, and Epstein, supra). Trabecular meshwork cells have been shown to possess actin and myosin filaments (Clark, et al., Invest. Ophthalmol. Vis. Sci., 35:281-294, 1994) and to contract in response to some agents (Coroneo, et al, Exp. Eye Res., 52:375-388, 1990; Lepple-Wienhues, et al., Exp. Eye Res., 53:33-38, 1991; Wiederholt, et al., Invest. Ophthalmol. Vis. Sci., 35:2515-2520, 1994). In a review of the existing literature at the time, Davson speculated that changes in trabecular meshwork cell volume (as distinct from cell shape) may participate in the regulation of aqueous outflow facility (Davson, H., Physiology of the Eye, H. Davson, ed., 5th Ed., Macmillan Press, London, Chapter 1, pp. 9-81, 1990), but to date this hypothesis has not been specifically addressed by other investigators. An excellent review of trabecular meshwork physiology and morphology is found in P. L. Kaufman, xe2x80x9cPressure-dependent Outflowxe2x80x9d in R. Pitch et al., ed. The Glaucomas, St. Louis, Mo.; C. V. Mosby Co., 1989, 219-240, Vol. 1.
In addition to regulation of aqueous outflow, trabecular meshwork cells are thought to serve an immunologic function as they phagocytize antigens in the anterior chamber of the eye as they pass through the trabecular meshwork (Epstein, et al., Invest. Opthalmol. Vis. Sci., 27:387-395, 1986). It has been hypothesized that the cells then migrate out of the meshwork into the Canal of Schlemm to enter the systemic circulation and act as antigen presenting cells to trigger the production of antibodies to the phagocytized antigen. In at least one form of glaucoma (pigmentary), this phagocytotic function is thought to be overwhelmed, resulting in increased resistance to aqueous outflow (Epstein, et al., supra). The endothelial cells lining the Canal of Schlemm also appear to contribute to the resistance to outflow in the normal eye (Davson, H., supra; Hart, W. M., supra).
A number of hormones and neurotransmitters have been documented to decrease intra-ocular pressure by modulating aqueous production or outflow. Studies employing a human eye perfusion model have shown that epinephrine, via an apparent xcex2-adrenergic effect upon the uveo-scleral pathway, increases the facility of outflow (Erickson-Lamy and Nathanson, supra). Nitrovasodilators have been found to increase outflow facility and decrease intra-ocular pressure in monkey eye (Schuman, et al., Exp. Eye Res., 58:99-105, 1994). Similarly, atrial natriuretic peptide decreases intra-ocular pressure in monkey eyes and increases aqueous humor production (Samuelsson-Almen, et al, Exp. Eye Res., 53:253-260, 1991). In addition to these hormones and neurotransmitters, ethacrynic acid has been shown to increase aqueous outflow and decrease intra-ocular pressure by modulating aqueous inflow and outflow. Elevations of norepinephrine concentration in the aqueous humor resulting from cervical sympathetic nerve stimulation cause an increase in intra-ocular pressure of rabbit eye in situ by a mechanism that appears to involve an a-adrenergic effect (Gallar, et al., Invest. Ophthalmol Vis. Sci., 34:596-605, 1993). Similarly, topical administration of vasopressin to the eye has been shown to increase intra-ocular pressure and decrease facility of outflow in both normal and glaucomatous human eyes (Becker, et al. Arch. Ophthalmol., 56: 1, 1956; Viggiano, et al., Am. J. Ophthalmol., 115: 511-516, 1993). A local renin-angiotensin system resides in the eye, and inhibition of angiotensin converting enzyme causes a decrease of intra-ocular pressure (Abrahms, et al., J. Ocular Pharmacol., 7:41-51, 1991; Deinum, et al., Endocrinol., 126:1673-1682, 1990). In contrast to these rapidly-acting agents, administration of the glucocorticoid dexamethasone increases resistance to outflow over a slower time course of hours and days, an effect that has been postulated to occur in the expression of extracellular matrix (Becker, et al., Arch. Ophthalmol., 70:500-507, 1963; Clark, et al., supra; Partridge, et al., supra; Polansky, et al., supra).
Relatively little is known about the signal transduction and ion transport properties of TM cells. Cultured bovine trabecular meshwork cells have been examined for their ability to regulate intracellular pH (Chu et al, Acta Ophthalmol, 70:772-779, 1992). These studies demonstrated that the cells possess a Na/H exchanger that is activated by intracellular acidification and inhibited by amiloride, as is Na/H exchange of other cell types. In other studies of cultured bovine TM cells, Coroneo, et al.,supra, have provided electrophysiological evidence that these cells also possess Na/K ATPase and K channels. The presence of Ca channels in these TM cells has been indicated by the observation that the Ca channel blocker nifedipine prevents endothelin-evoked depolarization of the cells (Lepple-Wienhues, et al., German J. Ophthalmol., 1:159-163, 1992). In addition, both plasma membrane and sarcolemmal Ca ATPases have been identified in rabbit TM cells by cytochemical methods (Kobayashi, et al., Acta Soc. Ophthalmol. Jap., 93:396-403, 1989). Naxe2x80x94Kxe2x80x94Cl cotransport is a plasma membrane ion transport system found in a wide variety of cell types, both epithelial and non-epithelial (Chipperfield, A., Clin. Sci., 71: 465-476, 1986; Haas. M., Ann. Rev. Physiol., 51:443-457, 1989; Pewitt. et al., J. Biol. Chem., 265(34):20747-20756, 1990). It is a bidirectional symport mechanism, indicating that each transport molecule binds to the three transported ion species (Na, K and Cl), and moves them together across the plasma membrane in the same direction. The transporter is bidirectional such that it can operate to move the ions into or out of the cell with the net direction of flux determined by the electrochemical gradients of Na, K and Cl. In many cells, the inwardly directed Na gradient is the most prominent, and net movement of these ions is directed into the cell (Chipperfield, A., supra; Haas, M., supra; O""Grady, et al., Am. J. Physiol., 253 :C177-C192, 1987). Other characteristic features of Naxe2x80x94Kxe2x80x94Cl cotransport include: 1) a high ion selectivity for Na, K and Cl; 2) an absolute requirement for the presence of all three ion species to operate; and 3) specific inhibition by xe2x80x9cloopxe2x80x9d diuretics (Palfrey, et al., supra).
There are two types of Naxe2x80x94Kxe2x80x94Cl cotransporters with different electroneutral stoichiometries. For most cells in which it has been studied (Ehrlich ascites tumor cells, rabbit kidney cells and duck red blood cells), the stoichiometry of cotransport is 1 Na+:K+:2Clxe2x88x92, but in Squid axon the stoichiometry is different, 2 Na+:1 K+:3 Cl. Whereas the kinetic and pharmacological features of Naxe2x80x94Kxe2x80x94Cl cotransport are quite constant among different cell types, the regulation of cotransport is heterogeneous. Elevation of intracellular cyclic AMP stimulates cotransport in some cells, while it inhibits cotransport in other cells. Similarly, elevation of cyclic GMP can have either stimulatory or inhibitory effects, and cotransport can be regulated by Ca and by phorbol esters, activators of protein kinase C (Chipperfield, A., supra; O""Donnell, et al., Proc. Natl. Acad. Sci., USA, 83:6132-6136, 1986; O""Donnell, et al., Am. J. Physiol., 255:C169-C180, 1988; Grady. et al., supra) For instance, in cultured vascular endothelial cells, Na+xe2x80x94K+xe2x80x942Clxe2x88x92 cotransport is inhibited by elevations of intracellular cyclic AMP and cyclic GMP, and by activation of protein kinase C. In contrast, elevation of intracellular Ca stimulates endothelial cell Na+xe2x80x94K+xe2x80x942Clxe2x88x92 cotransport (O""Donnell, M. E. Am. J. Physiol, 257:C36-C-44, 1989; O""Donnell, M. E., J. Biol. Chem., 266:11559-11566, 1991). The reports of several studies have suggested that regulation of cotransport activity by vasoactive agonists and by extracellular tonicity involve a direct phosphorylation of the cotransporter (Lyle, et al., J. Biol. Chem., 267:25428-25437, 1992; Pewitt, et al., supra; Torchia, et al., J. Biol. Chem., 267:25444-25450, 1992).
Two primary physiological functions have been demonstrated for Naxe2x80x94Kxe2x80x94Cl cotransport. The cotransporter participates in vectorial transport of ions across some epithelia, working in conjunction with the Na/K pump and other transport systems for Na, K and Cl (O""Grady, et al., supra). This function has been reported to occur in ciliary epithelial cells in the eye (Dong, et al., supra). The cotransporter also functions to regulate cell volume in a number of cell types, both epithelial and non-epithelial, in response to varying extracellular osmolarity (Eveloff, et al., Am. J. Physiol., 252:F1-F10, 1987; Kobayashi, et al., supra, MacKnight, A. D. C., Renal Physiol. Biochem., 3-5:114-141, 1988; O""Donnell, M. E., Am. J. Physiol., 264:1316-1326, 1993; O""Grady, et al., supra). When cells are exposed to hypertonic media, they shrink rapidly as water exits the cell down its concentration gradient. In cells that utilize Naxe2x80x94Kxe2x80x94Cl cotransport to regulate volume, the shrinkage of cells activates the cotransporter, which in turn mediates a net uptake of Na, K and Cl into the cell. As water re-enters the cell with the transported ions, the cell swells again. The Naxe2x80x94Kxe2x80x94Cl cotransport system performs this function in a number of cell types, including vascular endothelial cells, avian erythrocytes, Ehrlich ascites tumor cells, human fibroblasts, chick cardiac cells, and cells of rabbit renal thick ascending limb (Kregenow, F. M., Ann. Rev. Physiol., 4: 493-505, 1981; MacKnight, A. D. C., supra; O""Donnell, M. E., supra, 1993). Trabecular meshwork and vascular endothelium both present highly regulated barriers to solute and water flux.
A regulatory volume increase can also be mediated in some cell types by combined actions of the Na/H exchange Cl/HCO3 exchange systems (Kregenow, F. M., supra; MacKnight, A. D. C., supra). Exposure of cells to hypotonic media causes cells to swell rapidly as water enters the intracellular space, followed by a compensatory decrease in cell volume. The regulatory volume decrease appears to be mediated by a net efflux of ions through transporters separate from the Naxe2x80x94Kxe2x80x94Cl cotransporter, for example the K and Cl conductive pathways (Eveloff, et al., supra; Kregenow, F. M., supra; MacKnight, A. D. C., supra). In addition, vasoactive agents have been shown to modulate the cell volume regulating system of avian erythrocytes. In these cells, the volume set point appears to be increased by catecholamines, such that the response of the Naxe2x80x94Kxe2x80x94Cl cotransporter to extracellular tonicity is altered (Geck, et al., J. Memb. Biol., 91:97-105, 1986).
The glaucomas comprise a heterogeneous group of eye diseases in which elevated IOP causes damage and atrophy of the optic nerve, resulting in vision loss. The underlying cause of the elevated IOP can be grossly divided into two pathophysiologic scenarios in which the drainage pathways are either physically closed off (as in the various forms of angle-closure glaucoma) or in which the drainage pathways appear anatomically normal but are physiologically dysfunctional (as in the various forms of open-angle glaucoma). Angle-closure glaucoma is nearly always a medical and/or surgical emergency, in which pharmacologic intervention is essential in controlling an acute attack, but in which the long-range management is usually surgical Primary Open Angle Glaucoma (POAG), on the other hand, has a gradual, symptomless onset and is usually treated with chronic drug therapy. POAG is the most common form of glaucoma, comprising≈80% of newly-diagnosed cases in the USA, and is the leading cause of blindness among African Americans.
Drugs currently used to treat glaucoma can be divided into those that reduce aqueous humor inflow and those that enhance aqueous humor outflow. The most commonly-prescribed drugs at present are the xcex2-adrenergic antagonists, which reduce aqueous humor inflow through an unknown effect on the ciliary body. Other drugs that reduce aqueous inflow include inhibitors of carbonic anhydrase (e.g., acetazolamide and methazolamide) and the alpha-adrenergic agonist apraclonidine; both of these drug classes exert their clinical effects through a poorly-understood action on the ciliary body. Each of these drugs, although effective in many patients, is poorly tolerated in some because of profound and occasionally life-threatening systemic adverse effects.
Drugs that enhance aqueous humor outflow from the eye include miotics and the adrenergic agonists. The miotics exert a mechanical effect on the longitudinal muscle of the ciliary body and thus pull open the trabecular meshwork; they comprise both direct-acting parasympathomimetic agents (e.g., pilocarpine and carbachol) and indirect-acting parasympathomimetic agents (e.g., echothiopate). Miotic agents are highly effective in lowering IOP but have significant adverse effects, including chronic miosis, decreased visual acuity, painful accomodative spasm and risk of retinal detachment. Adrenergic agonists (e.g., epinephrine and dipivefrin) act on the uveoscleral outflow tract to enhance outflow through a mechanism that remains poorly understood. These drugs have perhaps the best safety profile of the compounds presently used to treat glaucoma but are among the least effective in their IOP-lowering effect.
Accordingly, the need exists for new and better methods of lowering intra-ocular pressure, particularly in the treatment of one of the leading causes of blindness, glaucoma.
The present invention provides a method for reducing intra-ocular pressure using a new class of compounds hitherto used as high ceiling diuretics, also known as loop diuretics, due to their ability to completely inhibit sodium chloride transport in the thick ascending limb of the loop of Henle. It has been discovered that the trabecular meshwork cells of the mammalian eye regulate cell volume and fluid transport by means of a Na+xe2x80x94K+xe2x80x942Clxe2x88x92 cotransporter mechanism, such as that found in the renal thick ascending limb of the loop of Henle. Compounds that substantially inhibit operation of this mechanism also increase the outflow of ocular fluids, thus lowering intra-ocular pressure. Such compounds are useful in treatment of diseases of the eye associated with elevated intra-ocular pressure, such as ocular hypertension and glaucoma.
Preferred compounds useful in the practice of this invention are furosemide, piretanide, benzmetanide, bumetanide, and torasemide, derivatives thereof with lipophilic and amphipathic characteristics designed to enhance penetration through the intact cornea, and pharmaceutically acceptable salts thereof. These outflow-increasing compounds are administered directly to the eye, either topically, by corneal iontophoresis, or by intracameral microinjection into the anterior chamber of the eye. The delivery of these compounds may be enhanced by the use of an erodible or sustained release ocular insert device. Lipophilic or amphipathic derivatives of these compounds are particularly preferred for topical administration.
A screening method is also provided to discover additional compounds with utility for lowering intra-ocular pressure by substantially inhibiting the Na+xe2x80x94K+xe2x80x942Clxe2x88x92 cotransporter mechanism in trabecular meshwork cells.